The Syrian golden hamster is the classic animal model for the in vivo study of respiratory carcinogenesis, Explant cultures or respiratory epithelial have been used in the past to study in vitro carcinogenesis, usually with serum-containing media. Because of the advantages of using serum-free media, the efficiency of various formulations for the explant culture of hamster respiratory epithelia was explored. The following media were tested: (1) Ham's F-12, (2) CMRL-1066, (3) Eagle's MEM, (4) MCDB 152, (5) Eagle's MEM without calcium with nonessential amino acids, (6) CMRL-1066 and Eagle's MEM without calcium with nonessential amino acids (1:1), and (7) Eagle's MEM with citrulline substituted for arginine. Each medium was tested either with 1% heat inactivated fetal bovine serum or with seven growth factors (insulin, hydrocortisone, ethanolamine, phosphoethanolamine, transferrin, epidermal growth factor, and bovine pituitary extract). The addition of serum resulted in a massive overgrowth of fibroblasts. Serum-free Ham's F-12 and CMRL-1066 supported the outgrowth of differentiated epithelial cells and retarded the growth of fibroblasts. In the case of tracheal epithelial cell suspensions isolated by protease digestion, F-12 supplemented with the above seven factors supported both growth and subculture. Coating of Petri dishes with a mixture of collagen, fibronectin and bovine serum albumin enhanced attachment of the isolated tracheal cells. Studies are under way to characterize these cells by morphological and biochemical techniques and to use them in chemically-induced transformation studies.